The sequencing of the human genome has allowed the identification of a vast number of putative genes. However, the function of only a small number of these genes can be inferred from their primary sequences. New techniques and agents are needed to cope with the task of assigning functional roles to these gene products. This implies determination of how, when and where they are involved in specific biochemical pathways. Ideally, these techniques and agents will allow the rapid screening of substantial subsets of the sum of a genome's products. Some methods have been designed for broad and rapid screening, but they are generally limited to in vitro application and do not necessarily provide information that is relevant to the function of proteins in living cells. Visualizing and monitoring specific proteins, with minimal disruption of their biological function and distribution, remains one of the foremost challenges in chemical biology. More powerful methods of detection of specific proteins and monitoring their localization and interactions inside living cells are urgently required.
One of the most widely applied methods for studying the expression, localization and trafficking of cellular proteins is the fluorescent labelling of a specific protein of interest (POI). This can be accomplished by genetically fusing the POI to an intrinsically fluorescent protein, or to an enzyme that can be labelled with a fluorescent inhibitor. However, the significant size of these fusion proteins can alter the biological function of the POI. Alternatively, a POI can be fused to a ‘substrate tag’ that can be site-specifically labelled through a subsequent enzymatic reaction. In this approach, the smaller size of the tag poses less risk for steric perturbation than the fusion of an entire protein; however, native enzymatic reactions can prove to be problematic for some cellular applications.
Maleimide groups have long been used in applications that exploit their propensity to react selectively with thiol groups, undergoing Michael addition reactions through their C2=C3 double bond (Kanaoka, Y. et al., Chem. Pharm. Bull. 1964, 12, 127). Maleimides are also known to quench fluorescence, probably due to their participation in a photoinduced electron transfer (PeT), allowing non-radiative relaxation of the fluorophore's excited state. The thiol addition reaction breaks the conjugation of the maleimide group, altering the energy levels of its molecular orbitals and removing its capacity to quench fluorescence (Guy, J. et al., J. Am. Chem. Soc. 2007, 129, 11969). These properties were demonstrated in the characterization of a naphthopyranone derivative bearing a maleimide group whose fluorescence increased dramatically upon reaction with glutathione (Langmuir, M. E. et al., Tetrahedron Lett. 1995, 36, 3989).
Labelling techniques based on the use of fluorescent dyes bearing reactive functional groups like maleimides, known to react with thiols, have been described (Tsien, R. Y., Annu. Rev. Biochem. 1998, 67, 509-544). However, these methods are typically non-specific, labelling the surface-exposed functional groups of many different proteins. Based on this chemical reaction, we previously developed a complementary labelling strategy based on the Fluorogenic Addition Reaction (FlARe) between a small, genetically encoded dicysteine peptide tag and dimaleimide fluorogenic labelling agents. In the FlARe approach to protein labelling, a POI is genetically fused to a short peptide tag (dC10α) presenting two Cys residues that are separated by two turns of the α-helical secondary structure of the tag, placing them ca. 10 Å apart. Fluorogenic labelling agents have also been designed, presenting two maleimide groups separated by ca. 10 Å that can react with the thiol groups of the dC10α tag sequence. The dimaleimide moiety quenches the fluorescence of the pendant fluorophore through a PeT mechanism, until both maleimide groups undergo thiol addition. The addition reactions restore the latent fluorescence and result in robust, covalent labelling (Keillor, J. W. et al., Org Biomol Chem 2011, 9, 185-197; Keillor, J. W. et al., Mol Biosyst 2010, 6, 976-987; Keillor, J. W. et al., J Am Chem Soc 2007, 129, 11969-11977; Girouard, S. et al., J Am Chem Soc 2005, 127, 559-566). Using the FlARe approach, we were able to selectively label the POI.
Recently we refined the design of the dimaleimide fluorogens to enhance quench efficiency and the reactivity of the maleimide group, as demonstrated through selective no-wash intracellular labelling (Caron, K. et al., Org Biomol Chem 2011, 9, 185-197; Chen, Y. et al., Angew Chem Int Ed Engl 2014). However, known fluorogens include dansyl and coumarin groups, both of which require excitation with UV or blue light, raising questions regarding the risk of photodamage. There is a need for fluorogens with longer excitation wavelength that can be visualized in the green and red channels of fluorescence microscopes for use in FlARe labelling strategies.